Treatment of t-cell mediated diseases

ABSTRACT

The invention provides a method of treating T-cell mediated diseases and a method of inhibiting the activation of T-cells using certain diketopiperazines. The invention also provides methods of synthesizing diketopiperazines and pharmaceutical compositions comprising certain diketopiperazines. The invention further provides methods of making improved pharmaceutical compositions of proteins and peptides by either increasing or decreasing the content of diketopiperazines in the compositions and the resultant improved pharmaceutical compositions.

This application is a continuation of U.S. application Ser. No.12/753,671, filed Apr. 2, 2010, which is a continuation of U.S.application Ser. No. 10/846,482, filed May 14, 2004, now U.S. Pat. No.7,732,403 which claims benefit of provisional application Nos.60/471,017, filed May 15, 2003; 60/489,270, filed Jul. 21, 2003;60/514,930, filed Oct. 27, 2003; and 60/517,338, filed Nov. 4, 2003, thecomplete disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the treatment of T-cell mediated diseases andto the inhibition of the activation of T-cells using certaindiketopiperazines. The invention also relates to pharmaceuticalcompositions comprising certain diketopiperazines and to methods ofsynthesizing diketopiperazines. The invention further relates to methodsof making improved pharmaceutical compositions of proteins and peptidesto either increase or decrease the content of diketopiperazines in thecompositions and to the resultant improved pharmaceutical compositions.

BACKGROUND

T-cell mediated diseases represent a large number of immune systemdisorders. In particular, T-cells are thought to be the cells that startand perpetuate autoimmune diseases. Autoimmune diseases are a group ofeighty serious, chronic illnesses that afflict millions of people in theUnited States alone. Autoimmune diseases are characterized by reactivityof the immune system to endogenous (self) antigens. These immuneresponses to self antigens are maintained by the persistent or recurrentactivation of self-reactive T-cells and, directly or indirectly, theself-reactive T-cells are responsible for the characteristic tissueinjury and destruction seen in autoimmune diseases. Although manytreatments for autoimmune diseases and other T-cell mediated diseaseshave been proposed, there is still a need for additional treatments.

SUMMARY OF THE INVENTION

The present invention provides a method of treating T-cell mediateddiseases. The method comprises administering to an animal in needthereof an effective amount of a diketopiperazine having the followingformula:

wherein:

-   -   R¹ and R², which may be the same or different, each is:    -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine; provided, however, that when R¹ is the side chain of        asparagine or glutamine, then R² cannot be the side chain of        lysine or ornithine, and when R¹ is the side chain of lysine or        ornithine, then R² cannot be the side chain of asparagine or        glutamine;    -   (b) R¹ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline        and/or R² is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline; or    -   (c) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen; or            a physiologically-acceptable salt thereof.

The invention also provides a method of inhibiting the activation ofT-cells. The method comprises administering to an animal in need thereofan effective amount of a diketopiperazine of formula I or aphysiologically-acceptable salt thereof.

The invention further provides a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a diketopiperazine having thefollowing formula:

wherein:

-   -   R⁵ and R⁶, which may be the same or different, each is:    -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine or ornithine; provided, however, that when R⁵ is the        side chain of asparagine or glutamine, then R⁶ cannot be the        side chain of lysine or ornithine, and when R⁵ is the side chain        of lysine or ornithine, then R⁶ cannot be the side chain of        asparagine or glutamine;    -   (b) R⁵ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline        and/or R⁶ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline; or    -   (c) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (iv) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH group;            and/or        -   (v) an H which is attached to a carbon atom replaced by a            halogen; or            a physiologically-acceptable salt thereof.

The invention provides another method of treating a T-cell mediateddisease. The method comprises administering to an animal in need thereofan effective amount of a pharmaceutical composition comprising a proteinor peptide normally found in the animal, the protein or peptide havingbeen treated so that the composition also comprises at least onediketopiperazine derived from the protein or peptide.

The invention further provides a method of inhibiting T-cell activation.The method comprises administering to an animal in need thereof aneffective amount of a pharmaceutical composition comprising a protein orpeptide normally found in the animal, the protein or peptide having beentreated so that the composition also comprises at least onediketopiperazine derived from the protein or peptide.

In addition, the invention provides methods of synthesizingdiketopiperazines. In one embodiment, the method comprises heating asolution of a protein or peptide under conditions effective to cause theformation of a diketopiperazine. In a second embodiment, the methodcomprises contacting a solution of a protein or peptide with an enzymethat cleaves the two N-terminal or the two C-terminal amino acids of theprotein or peptide under conditions effective to produce adiketopiperazine.

The invention also provides an improved pharmaceutical composition of aprotein or peptide. The improvement is that the composition comprises adecreased content of diketopiperazines.

In addition, the invention provides a method of making an improvedpharmaceutical composition of a protein or peptide. The method comprisesremoving from the composition at least some of the diketopiperazinespresent in the composition.

The invention further provides a method of making an improvedpharmaceutical composition of a protein or peptide. The method comprisestreating a solution of the protein or peptide so as to increase thecontent of diketopiperazines in the composition.

The invention also provides an improved pharmaceutical composition of aprotein or peptide. The improvement is that the composition comprises anincreased content of diketopiperazines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Tracings of counts versus concentration of ERK1/2 for TriPScells (CD4+ T-cell line isolated from influenza-immunized donor which isspecific for hemagglutinin) isolated on day 20 after stimulation withanti-CD3 OKT3 antibody and incubated with 25 ng phorbal myristic acid(PMA), HC-RBL (fraction of heated human colostrum of molecular weightless than 3 kD and containing MR-DKP) at a 1:10 dilution and 0.5 mMDA-DKP for 15 minutes at 37° C.

FIG. 2. Bar graph showing inhibition of secretion of tumor necrosisfactor α (TNFα) and IL-16 from TriPS cells 12 days after stimulationwith anti-CD3 OKT3 antibody. Indicates the inhibition of both TNFα andIL-16 secretion by human colostrum (HC) 2626 (containing MR-DKP) bandDA-DKP. The maximal release observed using HC 2626 at 1:100 and 1:1000dilutions is due to the lytic effect of high concentrations of humancolostrum. No lysis is observed using 0.5 mM DA-DKP, and TNFα and IL-16secretion are decreased.

FIG. 3. Bar graph showing inhibition of TNFα secretion from TriPS cells10 days after stimulation with anti-CD3 OKT3 antibody. Indicates that HCRBL and DA-DKP need to be investigated further for titratable responseas seen with HC 2626. May indicated a potent activity.

FIG. 4. Bar graph showing inhibition of TNFα secretion from TriPS cellsat varying times after stimulation with anti-CD3 OKT3 antibody.Indicates that early in the stimulation cycle, the effect of DA-DKP andHC RBL is inhibitory, while later in the cycle (day 14) the effect isstimulatory. HC 2626 inhibits at all times, presumably due to otherconstituents.

FIG. 5. Bar graph showing inhibition of TNFα secretion from H4#9.25cells (CD4+ T-cell line isolated from autopsy brain tissue of a multiplesclerosis patient which is specific for myelin basic protein) on day7-10 after stimulation with anti-CD3 OKT3 antibody. Indicates that TNFαsecretion from this T-cell line is also inhibited by HC 2626, HC RBL andDA-DKP.

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS

The present invention provides a method of treating T-cell mediateddiseases. “Treat” is used herein to mean to reduce (wholly or partially)the symptoms, duration or severity of a disease, including curing thedisease, or to prevent the disease.

T-cell mediated diseases include graft rejection, graft versus hostdisease, unwanted delayed-type hypersensitivity reactions (such asdelayed-type allergic reactions), T-cell mediated pulmonary diseases,and autoimmune diseases. T-cell mediated pulmonary diseases includesarcoidosis, hypersensitivity pneumonitis, acute interstitialpneumonitis, alveolitis, pulmonary fibrosis, idiopathic pulmonaryfibrosis and other diseases characterized by inflammatory lung damage.Autoimmune diseases include multiple sclerosis, neuritis, polymyositis,psoriasis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, Type 1diabetes, autoimmune pancreatitis, inflammatory bowel diseases (e.g.,Crohn's disease and ulcerative colitis), celiac disease,glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroiddiseases (e.g., Hashimoto's thyroiditis and Graves disease), myastheniagravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris,primary biliary cirrhosis, pernicious anemia, and systemic lupuserythematosis.

The T-cell mediated disease are treated by administering to an animal inneed thereof an effective amount of a diketopiperazine having thefollowing formula:

wherein:

-   -   R¹ and R², which may be the same or different, each is:    -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine; provided, however, that when R¹ is the side chain of        asparagine or glutamine, then R² cannot be the side chain of        lysine or ornithine, and when R¹ is the side chain of lysine or        ornithine, then R² cannot be the side chain of asparagine or        glutamine;    -   (b) R¹ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline        and/or R² is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline; or    -   (c) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen; or            a physiologically-acceptable salt thereof.

By “replaced” is meant that, with reference to the formula of an aminoacid side chain, the specified group is replaced by the other specifiedgroup. For instance, the formula of the isoleucine side chain is—CH(CH₃)—CH₂—CH₃. If the terminal —CH₃ group is replaced with a —CH₂—OHgroup, then the formula of the resulting derivatized isoleucine sidechain would be —CH(CH₃)—CH₂—CH₂—OH. As another example, the formula ofthe alanine side chain is —CH₃. If one of the hydrogen atoms is replacedby a chlorine atom, then the resulting derivatized alanine side chainwould be —CH₂—Cl. Note that the side chain of glycine is —H and, if thisH is replaced by a chlorine (or other halogen) atom, the resulting sidechain will —Cl, with the chlorine atom attached to the ring carbon(e.g., R¹=—Cl)

Preferred are diketopiperazines wherein R¹, R² or both is the side chainof aspartic acid or glutamic acid or a derivative of such a side chainwherein the —COOH group is replaced by a —COOR³ group or a —CON(R⁴)₂group, wherein R³ and R⁴ are defined above. Of this group of compounds,most preferred are diketopiperazines comprising the side chains ofaspartic acid and alanine (Asp-Ala DKP or DA-DKP), the side chains ofglutamic acid and alanine (Glu-Ala DKP or EA-DKP), the side chains oftyrosine and aspartic acid (Tyr-Asp DKP or YD-DKP), the side chains oftyrosine and glutamic acid (Tyr-Glu DKP or YE-DKP) and derivatives ofthe aspartic acid or glutamic acid side chains of these fourdiketopiperazines wherein the —COOH group is replaced by a —COOR³ groupor a —CON(R⁴)₂ group, wherein R³ and R⁴ are defined above.

Also, preferred are diketopiperazines wherein R¹ and R² are bothhydrophobic side chains (e.g., the side chain of phenylalanine) orhydrophobic side chain derivatives. By “hydrophobic side chainderivative” is meant that the derivatized side chain which ishydrophobic. In particular, preferred are diketopiperzines wherein R¹and/or R², which may be the same or different, each is the side chain ofglycine, alanine, valine, norvaline, α-aminobutyric acid, leucine,isoleucine, norleucine or phenylalanine, and/or R¹ and/or R² is—CH₂—CH₂—CH₂— and together with the adjacent nitrogen atom(s) formproline. Of this group of compounds, most preferred are thediketopiperazines comprising the side chains of glycine and leucine(Gly-Leu DKP or GL-DKP), proline and phenylalanine (Pro-Phe DKP orPF-DKP), and alanine and proline (Ala-Pro DKP or AP-DKP)

Additional preferred diketopiperazines are those wherein R¹, R² or bothis the side chain of methionine, the side chain of arginine or aderivative of these side chains. Most preferred of this group is adiketopiperazine wherein R¹ is the side chain of methionine and R² isthe side chain of arginine (Met-Arg DKP or MR-DKP).

By “side chain” of an amino acid is meant that portion of the amino acidattached to the common NH₂—CH—COOH backbone of all of the amino acidslisted above. For instance, the side chain of glycine is —H, the sidechain of alanine is —CH₃, and the side chain of serine is —CH₂OH.

By “hydrophobic” is meant a side chain or side chain derivative that isuncharged at physiological pH and is repelled by an aqueous solution.

By “alkyl” is meant a saturated straight-chain or branched hydrocarboncontaining 1-10 carbon atoms, preferably 1-6, carbon atoms. “Loweralkyl” means a saturated straight-chain or branched hydrocarboncontaining 1-6 carbon atoms.

By “cycloalkyl” is meant a saturated cyclic hydrocarbon containing atleast one ring, each ring containing at least three carbon atoms.Preferably, the cycloalkyl contains one ring of 4-8 carbon atoms.

By “heterocycloalkyl” is meant a cycloalkyl having one or more of thering carbon atoms of at least one of the rings replaced by an O, S or N.

By “aryl” is meant an aromatic group having at least one aromatic ring(e.g., phenyl).

By “alkylaryl” is meant a lower alkyl having an H replaced by an aryl(e.g., —CH₂—C₆H₅ or —CH₃CH(C₆H₅)CH₃).

By “arylalkyl” is meant an aryl having an H replaced by a lower alkyl(e.g., —C₆H₄—CH₃).

By “heteroaryl” is meant an aryl having one or more of the ring carbonatoms of at least one of the rings replaced by an O, S or N.

By “substituted” is meant that the moiety is substituted with one ormore substituents selected from the following group: —OH, NH₂, —SH,—COOH and/or a halogen atom.

By “halogen” is meant chlorine, fluorine, bromine or iodine. Preferredis chlorine or bromine.

The diketopiperazines of formula I are effective in treating T-cellmediated diseases because they inhibit the activation of T-cells.Accordingly, the diketopiperazines of formula I can also be used totreat inflammation and inflammatory diseases which are caused by,exacerbated by, or involve activated T-cells. “Inhibit” is used hereinto mean to reduce (wholly or partially) or to prevent.

Methods of making diketopiperazines are well known in the art, and thesemethods may be employed to synthesize the diketopiperazines of theinvention. See, e.g., U.S. Pat. Nos. 4,694,081, 5,817,751, 5,990,112,5,932,579 and 6,555,543, US Patent Application Publication Number2004/0024180, PCT applications WO 96/00391 and WO 97/48685, and Smith etal., Bioorg. Med. Chem. Letters, 8, 2369-2374 (1998), the completedisclosures of which are incorporated herein by reference.

For instance, diketopiperazines can be prepared by first synthesizingdipeptides. The dipeptides can be synthesized by methods well known inthe art using L-amino acids, D-amino acids or a combination of D- andL-amino acids. Preferred are solid-phase peptide synthetic methods. Ofcourse, dipeptides are also available commercially from numeroussources, including DMI Synthesis Ltd., Cardiff, UK (custom synthesis),Sigma-Aldrich, St. Louis, Mo. (primarily custom synthesis), PhoenixPharmaceuticals, Inc., Belmont, Calif. (custom synthesis), FisherScientific (custom synthesis) and Advanced ChemTech, Louisville, Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determine by the person skilled in the art. Itwill advantageously be 2-9, preferably 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

To prepare the diketopiperazine of formulas I and II wherein the aminoacid side chains are derivatized, amino acid derivatives can be used inthe synthesis of the dipeptides, the dipeptides can be derivatizedand/or the diketopiperazines can be derivatized, as is known in the art.See, e.g., those references cited above.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above. In addition, many diketopiperazinessuitable for use in the present invention can be made as described belowfrom proteins and peptides. Further, diketopiperazines for use in thepractice of the invention can be obtained commercially from, e.g., DMISynthesis Ltd., Cardiff, UK (custom synthesis).

The diketopiperazines of formulas I and II include all possiblestereoisomers than can be obtained by varying the configuration of theindividual chiral centers, axes or surfaces. In other words, thediketopiperazines of formulas I and II include all possiblediastereomers, as well as all optical isomers (enantiomers).

The physiologically-acceptable salts of the diketopiperazines of theinvention may also be used in the practice of the invention.Physiologically-acceptable salts include conventional non-toxic salts,such as salts derived from inorganic acids (such as hydrochloric,hydrobromic, sulfuric, phosphoric, nitric, and the like), organic acids(such as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, glutamic, aspartic, benzoic, salicylic, oxalic,ascorbic acid, and the like) or bases (such as the hydroxide, carbonateor bicarbonate of a pharmaceutically-acceptable metal cation or organiccations derived from N,N-dibenzylethylenediamine, D-glucosamine, orethylenediamine). The salts are prepared in a conventional manner, e.g.,by neutralizing the free base form of the compound with an acid.

As noted above, a diketopiperazine of the invention, or aphysiologically-acceptable salt thereof, can be used to treat a T-cellmediated disease or to inhibit activation of T-cells. To do so, adiketopiperazine, or a physiologically-acceptable salt thereof, isadministered to an animal in need of treatment. Preferably, the animalis a mammal, such as a rabbit, goat, dog, cat, horse or human. Effectivedosage forms, modes of administration and dosage amounts for thecompounds of the invention may be determined empirically, and makingsuch determinations is within the skill of the art. It is understood bythose skilled in the art that the dosage amount will vary with theparticular compound employed, the disease or condition to be treated,the severity of the disease or condition, the route(s) ofadministration, the rate of excretion of the compound, the duration ofthe treatment, the identify of any other drugs being administered to theanimal, the age, size and species of the animal, and like factors knownin the medical and veterinary arts. In general, a suitable daily dose ofa compound of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect.However, the daily dosage will be determined by an attending physicianor veterinarian within the scope of sound medical judgment. If desired,the effective daily dose may be administered as two, three, four, five,six or more sub-doses, administered separately at appropriate intervalsthroughout the day. Administration of the compound should be continueduntil an acceptable response is achieved.

The compounds of the present invention (i.e., diketopiperazines andphysiologically-acceptable salts thereof) may be administered to ananimal patient for therapy by any suitable route of administration,including orally, nasally, rectally, vaginally, parenterally (e.g.,intravenously, intraspinally, intraperitoneally, subcutaneously, orintramuscularly), intracisternally, transdermally, intracranially,intracerebrally, and topically (including buccally and sublingually).The preferred routes of administration are orally and intravenously.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a compound or compounds of theinvention as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (i.e., one or more diketopiperazines of the inventionand/or physiologically-acceptable salts thereof) is mixed with one ormore pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monosterate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may be employedas fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of compoundsof the invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, drops and inhalants. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal or intraocular administration.For administration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more compounds of the invention and a suitable powder base,such as lactose or starch. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges, or, e.g.,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Typical ofatomizers are the Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with anaqueous or nonaqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be delivered bymeans of a simple eye dropper-capped bottle or by means of a plasticbottle adapted to deliver liquid contents dropwise by means of aspecially shaped closure.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

It has been found that diketopiperazines suitable for use in the presentinvention are present in some commercially-available intravenouspharmaceutical compositions containing albumin, immunoglobulin anderythropoietin. The diketopiperazines present in these pharmaceuticalpreparations are formed by the heating steps often used in themanufacture of these pharmaceutical compositions. The heating results incleavage and cyclization of the two N-terminal and/or two C-terminalamino acids of the proteins to form diketopiperazines.

Accordingly, diketopiperazines for use in the present invention can beprepared by heating solutions of albumin, immunoglobulin, erythropoietinand other proteins and peptides. For example, a solution of albumin,immunoglobulin, erythropoietin or another protein or peptide inphosphate buffer at neutral pH is prepared. Preferably, the solution isa concentrated solution (e.g., about 100-500 mM) to achieve protonationof the N-terminal and/or C-terminal amino acid. The solution is heatedat 60° C. for from about 2 hours to several days, preferably about 4days, to cause formation of the diketopiperazines. Denaturation of theprotein should, preferably, be avoided. This can be accomplished byusing shorter times and/or by adding caprylic acid or N-acetyltryptophan at about 0.02 M for each.

Diketopiperazines for use in the present invention can also be preparedby contacting a solution of albumin, immunoglobulin, erythropoietin oranother protein or peptide with an enzyme that can cleave the twoN-terminal amino acids from the protein or peptide (e.g., dipeptidylpeptidases) or an enzyme that can cleave the two C-terminal amino acidsfrom the protein or peptide (e.g., carboxypeptidases). Suitabledipeptidyl peptidases and carboxypeptidases are available commerciallyfrom, e.g., Sigma. The reaction should be conducted at pH 6-8,preferably in a buffer, such as phosphate buffer, at a temperature highenough to speed the reaction but not so high that the protein isdenatured (e.g., 37° C.).

The amino acid sequences of numerous proteins and peptides are known,and a protein or peptide with the desired N-terminal and/or C-terminalsequence can be chosen to give the desired diketopiperazine(s) usingeither method. Also, peptides with a desired sequence can be synthesizedby well known methods and used.

The diketopiperazines can be purified from solutions containing them,including from the commercially-available pharmaceutical compositionscomprising albumin, immunoglobulin and erythropoietin, by well knownmethods, such as size-exclusion chromatography (e.g., Centriconfiltration), affinity chromatography (e.g., using a column of beadshaving attached thereto an antibody or antibodies directed to thedesired diketopiperazine(s) or an antibody or antibodies directed to thetruncated protein or peptide), anion exchange or cation exchange. Thepurified diketopiperazines can be used and incorporated intopharmaceutical compositions as described above.

Instead of purifying the diketopiperazines, pharmaceutical compositionscomprising albumin, immunoglobulin, erythropoietin and/or other proteinsand/or peptides normally found in the animal recipient can beadministered for treatment of a T-cell mediated disease and can be usedto inhibit T-cell activation. Although compositions comprising theseproteins and/or peptides which are currently available commercially canbe used if they contain diketopiperazines, it is highly preferred totreat the albumin, immunoglobulin, erythropoietin and/or other proteinsand/or peptides as described above to increase the content of thedesired diketopiperzine(s) before administration of the thus improvedcompositions. The animal is preferably a human, and the proteins and/orpeptides are preferably human proteins and/or peptides. Oraladministration of the composition(s) is preferred.

Effective dosage amounts of the protein and/or peptide compositions canbe determined empirically, and making such determinations is within theskill of the art. In particular, to determine an effective dosage amountof a protein and/or peptide composition, the quantity of one or morediketopiperazines present in the composition can be measured, and anamount of the composition sufficient to deliver an effective amount ofthe diketopiperazine(s) can be administered to the animal. It isunderstood by those skilled in the art that the dosage amount will varywith the particular composition employed, the disease or condition to betreated, the severity of the disease or condition, the route(s) ofadministration, the rate of excretion, the duration of the treatment,the identify of any other drugs being administered to the animal, theage, size and species of the animal, and like factors known in themedical and veterinary arts. In general, a suitable daily dose of aprotein and/or peptide composition will be that amount which is thelowest dose effective to produce a therapeutic effect. However, thedaily dosage will be determined by an attending physician orveterinarian within the scope of sound medical judgment. If desired, theeffective daily dose may be administered as two, three, four, five, sixor more sub-doses, administered separately at appropriate intervalsthroughout the day. Administration should be continued until anacceptable response is achieved.

As noted above, it has been found that diketopiperazines are found incommercially-available intravenous pharmaceutical compositions ofalbumin, immunoglobulin and erythropoietin where manufacture of thesecompositions involves one or more heating steps (e.g., forsterilization). Diketopiperazines are also probably present in otherpharmaceutical compositions of proteins and peptides where manufactureof the compositions involves heating steps. As described herein, manydiketopiperazines have the ability to inhibit T-cell activation. Thus,it may not be desirable to administer compositions of albumin,immunoglobulin, erythropoietin or other proteins or peptides containingdiketopiperazines to patients in many situations. For instance, albuminis often administered to patients suffering from trauma, immunoglobulinis often administered to patients suffering from infections or immunedeficiencies, and erythropoietin is administered to anemic cancer orchronically ill patients whose immune systems are often compromised.Accordingly, the invention provides a method of removing at least some,preferably substantially all, of the diketopiperazines from suchcompositions. The diketopiperazines may be removed as described above(e.g., by size-exclusion chromatography (e.g., Centricon filtration),affinity chromatography (e.g., using a column of beads having attachedthereto an antibody or antibodies directed to the desireddiketopiperazine(s) or an antibody or antibodies directed to thealbumin, immunoglobulin, erythropoietin or other protein or peptide),anion exchange or cation exchange) to produce improved compositions ofalbumin, immunoglobulin, erythropoietin and other proteins and peptides.

EXAMPLES Example 1 Absorption of Asp Ala DKP (DA-DKP) and Glu Ala DKP(EA-DKP) From Rat Intestine

The rat intestine from the pyloric sphincter to the rectum wasmarginally isolated and perfused via the mesenteric artery with anerythrocyte based perfusate containing bovine serum albumin. Theeffluent perfusate from the gut was collected by cannulation of theportal vein and re-circulated (after re-oxygenation). After anequilibration period, a solution (approximately 1 ml) containingapproximately 1 mg of Asp-Ala diketopiperazine (DA-DKP) or 1.4 mg ofGlu-Ala diketopiperazine (EA-DKP) was administered by injection into thelumen of the duodenum.

After dosing, serial samples of the perfusate were collected at timedintervals up to 2 hours past dosing. Those samples were centrifuged andthe plasmas assayed for both cyclic dipeptides by tandem liquidchromatography mass spectrometry (LC-MS).

The results showed that, after only 2 hours perfusion, the amounts ofDA-DKP and EA-DKP which had been absorbed from the gut lumen into thecirculation corresponded to 95% and 100% (actually 112%), respectively,of the dose administered.

Thus, both cyclic peptides are absorbed rapidly and efficiently from thegut lumen into blood, with no evidence of metabolism during transportacross the gut wall. Hence these potential therapeutics may be given bymouth.

The rapid absorption of unchanged DA-DKP and EA-DKP from thegastrointestinal track into the blood combined with the lack of firstpass hepatic clearance of both compounds in the isolated perfused ratliver (data not shown) shows that pre-systemic clearance is low.Consequently oral dosing will be an ideal route of administration.

Moreover, studies with isolated perfused rat kidney showed that, unlikemany straight chain peptides, which are extensively metabolized by renalpeptidases, the renal clearance of both cyclic dipeptides is relativelyslow.

Collectively this data suggests that a dosing regimen of low daily dosesof diketopiperazines is likely to be adequate for therapeutic purposes.

Preliminary pharmacokinetic data in rats after oral administration wereconsistent with the above for both cyclic dipeptides, with T_(max)values of 30-60 minutes and C_(max) values of 4-6 μg/ml (DA-DKP) and0.6-1.1 μg/ml (EA-DKP) after oral dosing at 1.1-3.7 mg/kg body weight(DA-DKP) and 1.5-4.8 mg/kg body weight (EA-DKP) (T_(max) is the timewhen the concentration reaches a maximum, and C_(max) is the maximumconcentration reached; both were calculated from a curve fit equationfor the data obtained).

Preliminary data suggest that DA-DKP and other diketopiperazines crossthe blood-brain barrier. Thus, DA-DKP and other diketopiperazines of theinvention should be useful for treating nervous system disorders, suchas multiple sclerosis.

Example 2 Inhibition of Human T-Lymphocyte Cytokine Production In Vitroby Fractions of Human Colostrum Containing Met-Arg DKP (MR-DKP) and byAsp-Ala DKP (DA-DKP)

A. Materials

This example demonstrates that DA-DKP, human colostrum (HC 2626)containing MR-DKP, and a low-molecular weight fraction of humancolostrum (HC RBL; a fraction of human colostrum containing componentsof molecular weights less than 3000 prepared by Centricon filtration ofde-fatted colostrum) also containing MR-DKP, inhibited humanT-lymphocyte cytokine production. DA-DKP and MR-DKP were obtained fromDMI Synthesis, Ltd., Cardiff, UK. These two diketopiperazines are smallnaturally-occurring compounds generated during the physiologicalresponse to inflammation. They are also sometimes found in humanintravenous immunoglobulin (IVIg), human albumin and other biologicalpreparations.

B. Inhibition of T-Cell Cytokine Production

Two different CD4-positive human T-lymphocyte clones were tested. One ofthe cell lines (TRiPS) was isolated from an influenza-immunized donorand is specific for hemagglutinin peptide 307-319. The other cell line(H4#9.25) was isolated from the autopsy brain tissue of a multiplesclerosis donor and is specific for myelin basic protein (amino acids87-99). Both T-lymphocyte clones produce interleukin 8 (IL-8), IL-16,interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) afterin vitro stimulation with either (1) specific antigen plusHLA-DR2-positive presenting cells or (2) anti-CD3 plus anti-CD28antibodies.

The T-lymphocyte cell lines were stimulated for passage usingapproximately 4×10⁵ cells on day 18-20 after a previous stimulation.Cells were washed once in cold Iscove's Modified Dulbecco MinimalEssential Medium (IMDM, Sigma) plus 10% fetal bovine serum (FBS;American Type Culture Collection (ATCC)) and resuspended in 1.0 ml coldIMDM medium containing a 1:500 dilution of anti-CD3 monoclonal antibodyOKT3 (prepared from mouse ascites fluid). Cells were incubated withantibody for 30 minutes on ice, then washed with cold medium without FBSand combined with approximately 2×10⁶ 4000R-irradiated normal humandonor peripheral blood leukocytes (PBL), as feeder cells, in medium plus50 U/ml human IL-2 (Xenometrix). Cultures were expanded by the additionof fresh IMDM medium with FBS plus IL-2 on day 3. Day of culture ismeasured from the day of stimulation with OKT3. Cells can be used forexperiments starting on day 7 (at maximum proliferation), typically onday 14 (most sensitive to re-stimulation) and up until day 21 (restingcells approaching senescence).

Activation experiments were performed by withdrawing an aliquot of cellsand washing twice with warmed (37° C.) IMDM medium. For each specificassay, 2×10⁵ viable cells were pre-incubated in a total volume of 0.9 mlwarmed IMDM medium containing the specified amount of treatment additive(e.g., HC 2626, DA-DKP, PMA, etc.) for 15 minutes at 37° C. An aliquotof 2×10⁵ CD3/CD28 Dynabeads (Dynal), as activating stimulus, in 0.1 mlwarmed IMDM was then added and the cultures incubated overnight (18hours) at 37° C. Supernatants of the cell cultures were harvested afterpelleting the cells by centrifugation. Cytokine content was assayed byspecific ELISA (e.g., TNFα, IFNγ, IL-8, IL-16; Endogen).

As shown in FIGS. 1-5, human colostrum (HC 2626) inhibited the in vitrocytokine production by both of the T-lymphocyte cell lines in adose-dependent manner. As also shown in FIGS. 1-5, HC RBL and DA-DKPinhibited the in vitro cytokine production by both of the T-cell linesin a dose-dependent manner early in the stimulatory cycle. However, theeffects of HC RBL and DA-DKP later in the cycle (day 14 or later) werestimulatory (see FIG. 4). HC 2626 and HC RBL both contain MR-DKP (asdetermined by mass spectrometry), but HC 2626 contains otherconstituents (including caseins that are relatively dephosphorylatedproteins which may, therefore, be anti-inflammatory, as described inco-pending application Ser. No. 10/723,247, filed Nov. 25, 2003) besidesMR-DKP which may be responsible for its inhibitory effects later in thecell cycle. Accordingly, HC RBL and HC 2626 (both containing MR-DKP),MR-DKP and DA-DKP should be useful in down-modulating the inflammatorycytokine response in T-cell-mediated and/or autoimmune diseases, such asmultiple sclerosis, since they all inhibit cytokine production byT-cells early in the stimulatory cycle. These results also suggest thatHC RBL, HC 2626, MR-DKP and DA-DKP will selectively affectantigen-specific T-cells without affecting resting T-cells.

C. Mechanism of Action

The mechanism of action of DA-DKP and HC 2626 (containing MR-DKP) wasinvestigated. To do so, 1×10⁶ day 18 TRiPS cells were incubated for 30minutes at 37° C., either with nothing added (“Nil”), with CD3/CD28Dynabeads added (CD3/CD28 beads), with CD3/CD28 beads and 0.5 mM DA-DKP,or with CD3/CD28 beads and 1:500 dilution of HC 2626 added. After theincubation, the cells were lysed in Cell-Lytic Mammalian Cell ExtractionReagent (Sigma).

The cell extracts were then separately incubated with duplicateHypromatrix Arrays for 2 hours at room temperature, followed by twowashes following the manufacturer's (Hypromatrix) protocol. TheHypromatrix Array is a nylon membrane blotted with antibodies to thetranscription factors listed in Table 1 (custom manufactured byHypromatrix). An antibody cocktail specific for phosphorylated-tyrosine,phosphorylated-serine and phosphorylated-threonine (Zymed) was added,incubated for 1 hour. Then, an anti-immunoglobulin antibody labeled withbiotin was added. After washing the anti-immunoglobulin-biotin away,streptavidin-peroxidase was added, and the arrays given a final washbefore adding a peroxidase-reactive luminescent substrate.

The results were visualized by exposure to film and scored as 0(negative) or + to ++++ (positive) as presented in Table 2. As shown inTable 2, some cytokine transcriptional factor activation (ERK1/2) andrelease of pre-formed cytokines were inhibited by HC 2626 (containingMR-DKP) and DA-DKP.

TABLE 1 HYPROMATRIX ARRAY (CUSTOM): PROTEINS FOR PHOSPHORYLATION NUM-BER ACRONYM COMPOUND 1 Akt 1/2 protein kinase B, anti-apoptotic kinase 2c-Cbl TcR inhibitory pathway; Tyr²⁹² POation activates binding andinactivation of Syk and ZAP-70 3 CBP csk-binding protein (PAG); integralmembrane protein transiently (and at low level) Tyr-de-POated to releasecsk 4 CREB cAMP response element binding protein; POated (unk) toactivate/down-reg IL-2 promoter 5 csk COOH-terminal src kinase;Ser³⁶⁴-POated, also Tyr-POated (activity?) - POates and inactivates lck6 ERK1 extracellular signal-related kinase 7 c-fos AP-1 constituentactivated by TcR stimulation; POated at both N- and C-unk residues 8NFATC nuclear factor of activated T-cells; intact in anergy 9 c-jun AP-1constituent activated by TcR activation; POated by JNK-MAPK at Ser⁶³ 10IκB-α inhibitor of NFκB 11 pIκB-α Ser-POated and inactivated NFκBinhibitor 12 p38 MAPK mitogen-activated protein kinase 13 pI3 kinase/p85activated by glucocorticoids and β2- adrenergic-R 14 pten cytoplasmic3′-inositol phosphatase; tumor suppressor gene antagonizes PI 3′kinaseby converting PI-PO back to inactive forms 15 c-Raf-1 16 Rap1 negativeTcR regulatory GTPase 17 Ras kinase; inactivated during anergy 18 fyncell membrane-bound immediate TcR signal kinase 19 lck cellmembrane-bound immediate TcR signal kinase, active form is Tyr³⁹⁵POated; inactivated by csk POation at C-term Tyr 20 ZAP70kinasesignaller from CD3ζ; POated at ? by lck/fyn, ZAP70 POates LAT (linkerfor activation of T-cells) at Tyr's and Tyr's on SLP-76

TABLE 2 RESULTS COMPOUND NIL CD3/CD28 DKP HC2626 Akt 1/2 + ++ +++ ++c-Cbl — — — — CBP + ++ ++ ++ CREB — — — — csk + ++ + + ERK1 + + + +c-fos — — — — NFATC — — — — c-jun ++ + + + IκB-α ++ ++ + + pIκB-α — — —— p38 MAPK ++ +++ +++ +++ pI3 kinase/p85 + ++ + ++ pten — — — — c-Raf-1— — — — Rap1 + ++ ++ + Ras — — — — fyn + + + + lck — — — — ZAP70kinase —— — —

Example 3 Inhibition of Human T Lymphocyte Cytokine Production In Vitroby Gly-Leu DKP (GL-DKP) and Ala-Pro DKP (AP-DKP)

GL-DKP and AP-DKP (obtained from DMI Synthesis, Ltd., Cardiff, UK) weretested as described in Example 2 using TRiPS and H4#9.25 cell lines.GL-DKP and AP-DKP were found to inhibit the in vitro cytokine productionby both of these T-lymphocyte cell lines in a dose-dependent manner. Themechanism of action is currently under investigation as described inExample 2, and both cytokine transcriptional factor activation andrelease of pre-formed cytokine appear to be affected.

Example 4 Inhibition of Human T Lymphocyte Cytokine Production In Vitroby Asp Ala DKP (DA-DKP) and Tyr Glu DKP (YE-DKP)

Normal human lymphocytes were isolated from the peripheral blood of anormal human donor with Histopaque (Sigma). Then, 3-4×10⁵ of thelymphocytes were suspended in 1 ml of IMDM medium without serum. Thecells were stimulated with by adding 25 μl of a 1:2000 dilution ofanti-CD3 antibody (Pharmingen, San Diego, Calif.) and incubating for 18hours at 37° C.

Then, one of three DKP preparations and dexamethasone (finalconcentration of 10⁻⁵ M) were added to triplicate cultures. The threeDKP preparations were:

1. DA-DKP (obtained from DMI Synthesis, Ltd., Cardiff, UK; finalconcentration of 25 μg/ml in the cultures).

2. DKP-ZLB, a 25% albumin preparation (obtained from ZLB Bioplasma, AG3000 Berne 22 Switzerland) heated for 4 days at 60° C., after which itwas found to contain 0.5 mM DA-DKP, as determined by mass spectrometry(final concentration of 14 μg/ml DA-DKP in the cultures).

3. DKP-γ-glob—a γ-globulin preparation (obtained from Sigma, numberG-4386) containing 12 mg/ml γ-globulin in phosphate-buffered saline, pH7.4, was filtered using a Centricon 3000 filter, and the filtrate(containing components having MW less than 3000) was used. The filtratecontained a mass of 292, which is the mass of Tyr-Glu DKP (YE-DKP), asdetermined by anion exchange HPLC coupled to negative electrospray massspectrometry. The filtrate was used at a 1:4 final dilution in thecultures.

After addition of the DKP preparations or dexamethasone, the cultureswere incubated for 18 hours at 37° C. Then, the amounts of IL-2, IFNγand TNFα released into each culture were measured by ELISA (PierceBiotechnology, Rockford, Ill. 61105).

The results are presented in Table 3 below. As can be seen, the greatestreduction of release of all three cytokines was obtained withDKP-γ-glob. Flow cytometry looking at the number of CD69+ T-cells (CD69is a marker found on activated T-cells) also showed that DKP-γ-globreduced the number of CD69+ T-cells by about 90%, as compared to areduction of about 50% by dexamethasone, despite the internalization ofT-cell receptor complex.

TABLE 3 Stimulation Treatment U/ml IL-2 pg/ml IFNγ pg/ml TNFα Nil — 0.24± 0.1   2.3 ± 0.9 2.8 ± 0.5 CD3 — 2.6 ± 0.5 289 ± 35  98 ± 3.2 CD3DA-DKP 1.4 ± 0.3 306 ± 17  74 ± 4.7 CD3 DKP-ZLB 1.4 ± 0.4 311 ± 18 130 ±2.9  CD3 DKP-γ-glob 0.24 ± 0.25  2.1 ± 0.1 1.6 ± 0.6 (91% (99% (98%reduction) reduction) reduction) CD3 Dexamethasone 0.9 ± 0.1   76 ± 7.324.1 ± 0.3 (65% (74% (96% reduction) reduction) reduction)

1-91. (canceled)
 92. A method of treating inflammation or aninflammatory disease involving, caused by or exacerbated by T-cells,comprising administering to an animal in need thereof an effectiveamount of a pharmaceutical composition comprising a filtrate of asolution of a protein or peptide, wherein the filtrate is produced bypassing the solution of a protein or peptide over an ultrafiltrationmembrane with a molecular weight cutoff that retains a protein selectedfrom the group consisting of albumin, immunoglobulin and erythropoietin,and wherein the filtrate comprises: a diketopiperazine selected from thegroup consisting of YE-DKP, MR-DKP and DA-DKP.
 93. The method of claim92 wherein the filtrate contains components having molecular weightsless than
 3000. 94. The method of claim 92 wherein the protein orpeptide is a human protein or peptide.
 95. The method of claim 92wherein the protein or peptide is a human albumin.
 96. The method ofclaim 93 wherein the protein or peptide is a human protein or peptide.97. The method of claim 93 wherein the protein or peptide is a humanalbumin.
 98. The method of claim 92 wherein the animal is a human.
 99. Amethod of reducing the symptoms, severity or both of a T-cell mediateddisease, comprising administering to an animal in need thereof aneffective amount of a pharmaceutical composition comprising a filtrateof a solution of a protein or peptide, wherein the filtrate is producedby passing the solution of a protein or peptide over an ultrafiltrationmembrane with a molecular weight cutoff that retains a protein selectedfrom the group consisting of albumin, immunoglobulin and erythropoietin,and wherein the filtrate comprises: a diketopiperazine selected from thegroup consisting of YE-DKP, MR-DKP and DA-DKP.
 100. The method of claim99 wherein the filtrate contains components having molecular weightsless than
 3000. 101. The method of claim 99 wherein the protein orpeptide is a human protein or peptide.
 102. The method of claim 99wherein the protein or peptide is a human albumin.
 103. The method ofclaim 100 wherein the protein or peptide is a human protein or peptide.104. The method of claim 100 wherein the protein or peptide is a humanalbumin.
 105. The method of claim 99 wherein the animal is a human. 106.The method of claim 99 wherein the T-cell mediated disease is graftrejection, graft versus host disease, an unwanted delayed-typehypersensitivity reaction, a T-cell mediated pulmonary disease or anautoimmune disease.
 107. The method of claim 99 wherein the T-cellmediated disease is multiple sclerosis, neuritis, polymyositis,psoriasis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, Type 1diabetes, autoimmune pancreatitis, inflammatory bowel diseases, Crohn'sdisease, ulcerative colitis, celiac disease, glomerulonephritis,scleroderma, sarcoidosis, autoimmune thyroid diseases, Hashimoto'sthyroiditis, Graves disease, myasthenia gravis, Addison's disease,autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis,pernicious anemia, or systemic lupus erythematosis.
 108. The method ofclaim 99 wherein the T-cell mediated disease is pulmonary fibrosis oridiopathic pulmonary fibrosis.
 109. A method of treating inflammation oran inflammatory disease involving, caused by or exacerbated by T-cells,comprising administering to an animal in need thereof an effectiveamount of a pharmaceutical composition comprising an alanine-asparticacid diketopiperazine (DA-DKP) and a component selected from the groupconsisting of caprylic acid, N-acetyl tryptophan and combinationsthereof.
 110. The method of claim 109, wherein the pharmaceuticalcomposition is produced by passing a solution of albumin over anultrafiltration membrane with a molecular weight cutoff that retainsalbumin, and wherein the filtrate comprises the DA-DKP.
 111. The methodof claim 110, wherein the pharmaceutical composition filtrate containscomponents having molecular weights less than
 3000. 112. The compositionof claim 110, wherein the albumin is a human albumin.
 113. A method ofreducing the symptoms, severity or both of a T-cell mediated disease,comprising administering to an animal in need thereof an effectiveamount of a pharmaceutical composition comprising an alanine-asparticacid diketopiperazine (DA-DKP) and a component selected from the groupconsisting of caprylic acid, N-acetyl tryptophan and combinationsthereof.
 114. The method of claim 113, wherein the pharmaceuticalcomposition is produced by passing a solution of albumin over anultrafiltration membrane with a molecular weight cutoff that retainsalbumin, and wherein the filtrate comprises the DA-DKP.
 115. The methodof claim 114, wherein the pharmaceutical composition filtrate containscomponents having molecular weights less than
 3000. 116. The compositionof claim 114, wherein the albumin is a human albumin.